EP3013968B1 - Verfahren zur extraktion von öl aus dünnsilage - Google Patents

Verfahren zur extraktion von öl aus dünnsilage Download PDF

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Publication number
EP3013968B1
EP3013968B1 EP14817743.9A EP14817743A EP3013968B1 EP 3013968 B1 EP3013968 B1 EP 3013968B1 EP 14817743 A EP14817743 A EP 14817743A EP 3013968 B1 EP3013968 B1 EP 3013968B1
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Prior art keywords
protease
alpha
amylase
seq
starch
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EP3013968A4 (de
EP3013968A1 (de
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Nathanial KREEL
Joseph Jump
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Novozymes AS
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Novozymes AS
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B13/00—Recovery of fats, fatty oils or fatty acids from waste materials
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D3/00—Distillation or related exchange processes in which liquids are contacted with gaseous media, e.g. stripping
    • B01D3/001—Processes specially adapted for distillation or rectification of fermented solutions
    • B01D3/002—Processes specially adapted for distillation or rectification of fermented solutions by continuous methods
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/74—Recovery of fats, fatty oils, fatty acids or other fatty substances, e.g. lanolin or waxes

Definitions

  • the present invention relates to processes of extracting/recovering oil from thin stillage and/or syrup at the backend of a fermentation product production process based on starch-containing material.
  • Fermentation products such as ethanol are typically produced by first grinding starch-containing material in a dry-grind or wet-milling process, then degrading the material into fermentable sugars using enzymes and finally converting the sugars directly or indirectly into the desired fermentation product using a fermenting organism.
  • Liquid fermentation products are recovered from the fermented mash (often referred to as "beer mash"), e.g., by distillation, which separate the desired fermentation product from other liquids and/or solids.
  • the remaining fraction is referred to as "whole stillage”.
  • the whole stillage is dewatered and separated into a solid and a liquid phase, e.g., by centrifugation.
  • wet cake (or “wet grains") and the liquid phase (supernatant) is referred to as "thin stillage”.
  • Wet cake and thin stillage contain about 35 and 7% solids, respectively.
  • Dewatered wet cake is dried to provide "Distillers Dried Grains” (DDG) used as nutrient in animal feed.
  • DDG Disdens Dried Grains
  • Thin stillage is typically evaporated to provide condensate and syrup or may alternatively be recycled directly to the slurry tank as “backset”. Condensate may either be forwarded to a methanator before being discharged or may be recycled to the slurry tank.
  • the syrup may be blended into DDG or added to the wet cake before drying to produce DDGS (Distillers Dried Grain with Solubles).
  • DDGS Dangers Dried Grain with Solubles.
  • An increasing number of ethanol plants extract oil from the thin stillage and/or syrup/evaporated centrate as a by-product for use in biodiesel production or other biorenewable products.
  • WO 2011/126897 discloses processes of recovering oil by converting starch-containing materials into dextrins with alpha-amylase; saccharifying with a carbohydrate source generating enzyme to form sugars; fermenting the sugars using fermenting organism; wherein the fermentation medium comprises a hemicellulase; distilling the fermentation product to form whole stillage; separating the whole stillage into thin stillage and wet cake; and recovering oil from the thin stillage.
  • the fermentation medium may further comprise a protease.
  • WO 2012/088303 discloses processes for producing fermentation products, such as ethanol, including liquefying starch-containing material with a thermostable alpha-amylase and optionally a protease, e.g., one derived from Pyrococcus furiosus.
  • the object of the present invention is to provide improved processes of extracting or recovering oil at the backend of a fermentation product production process, such as especially an ethanol production process.
  • the invention relates to processes of recovering oil, comprising
  • the temperature in step (a) is above the initial gelatinization temperature, such as between 80-90°C, such as around 85°C.
  • Steps (b) and (c) may be carried out simultaneously or sequentially.
  • steps (a), (b) and (c) are carried our simultaneously or sequentially.
  • the temperature is below the initial gelatinization temperature, such as between 25-40°C, preferably around 32°C in case of producing fermentation products such as ethanol.
  • the protease is present in or added to the whole stillage in step (d) and/or to the thin stillage after separating whole stillage into thin stillage and wet cake in step (e), and/or to the syrup in step (e').
  • step (a) may be carried out at temperatures at or above the initial gelatinization temperature, such as between 80-90°C, or below the initial gelatinization temperature, such as between 25-40°C.
  • oil is recovered from the thin stillage and/or syrup/evaporated centrate, e.g., by extraction, such as hexane extraction or by using another oil recovery technology well-known in the art.
  • the protease may be any protease having at least 80% identity to SEQ ID NO: 4 and having a thermostability value, as defined herein, of more than 50% determined as Relative Activity. Determination of "Relative Activity” and "Remaining Activity” is determined as described in Example 1.
  • the protease has a thermostability value of more than 60%, more than 70%, more than 80%, more than 90% more than 100%, such as more that 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70°C.
  • the protease used in a process of the invention is a thermostable protease derived from the bacterium, e.g., classified as EC 3.4.21.62, such as Pyrococcus furiosus, such as the protease shown in SEQ ID NO: 4 or one having a sequence identity thereto of at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.
  • the protease used in a process of the invention has a thermostability value of more than 60%, more than 70%, more than 80%, more than 90% more than 100%, such as more that 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70°C for oil recovery from thin stillage and/or syrup at the backend of a fermentation product production process based on starch-containing material.
  • the object of the present invention is to provide improved processes of extracting or recovering oil at the backend of a fermentation product production process, such as especially an ethanol production process.
  • the invention relates to processes of recovering oil, comprising
  • step (a) is above the initial gelatinization temperature, such as between 80-90°C, such as around 85°C.
  • step (a) is carried out as a liquefaction step followed by steps (b) and (c) carried out either simultaneously or sequentially.
  • steps (b) and (c) are carried out as a simultaneous saccharification and fermentation step (i.e., SSF).
  • steps (a), (b), and (c) are carried out simultaneously. This is typically done at a temperature below the initial gelatinization temperature, i.e. raw starch hydrolysis process (RSH). However, steps (a), (b), and (c) may also be carried out sequentially. In such embodiments step (a) may be carried out at temperatures at or above the initial gelatinization temperature, such as between 80-90°C, or below the initial gelatinization temperature, such as between 25-40°C, such as around 32°C.
  • initial gelatinization temperature means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C., Starch/Stärke, Vol. 44 (12) pp. 461-466 (1992 ).
  • the protease is present and/or added at the backend of a fermentation product production process, such as an ethanol production process, preferably to the whole stillage and/or syrup/evaporated centrate, including a conventional ethanol product production process, which included a liquefaction step, e.g., step (a) done at high temperatures, such as at temperatures at or above the initial gelatinization temperatures, such as at temperatures between 80-90°C, at a pH between 4.5 and 6.0, followed by simultaneous saccharification and fermentation (e.g., steps (b) and (c)) done a temperature between 25-40°C, such as around 32°C, if the fermentation product is ethanol or the like.
  • a liquefaction step e.g., step (a) done at high temperatures, such as at temperatures at or above the initial gelatinization temperatures, such as at temperatures between 80-90°C, at a pH between 4.5 and 6.0
  • simultaneous saccharification and fermentation e.g., steps (b) and (c)
  • the protease is present and/or added at the backend of a fermentation product production process, such as an ethanol production process, preferably to the whole stillage and/or syrup, where granular starch is saccharified and fermented simultaneously at temperatures below the initial gelatinization temperatures, such as at temperatures between 25-40°C, at a pH between 4.5 and 6.0, i.e., steps (a) (b), and (c) are carried out simultaneously.
  • a fermentation product production process such as an ethanol production process
  • the protease is present in or added to the whole stillage in step (d) and/or to the thin stillage after separating the whole stillage into thin stillage and wet cake in step (e), and/or to the syrup in step (e').
  • oil is recovered from the thin stillage and/or syrup/evaporated centrate, e.g., by extraction, such as by hexane extraction or by using another oil recovery technology well-known in the art.
  • the protease has a thermostability value of more than 50%, more than 60%, more than 70%, more than 80%, more than 90% more than 100%, such as more that 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70°C.
  • the protease is a thermostable protease derived from the bacterium Pyrococcus furiosus, such as the protease shown in SEQ ID NO: 4, or one having sequence identity thereto of at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.
  • the Pyrococcus furiosus protease shown in SEQ ID NO: 4 herein is a thermostable bacterial protease.
  • a commercial Pyrococcus furiosus protease product (Pfu S) from Takara Bio Inc. (Japan) have been found to have a thermostability value of 110% (80°C/70°C) and 103% (90°C/70°C) at pH 4.5 determined as described in Example 1 herein.
  • the protease is a thermostable protease derived from the bacterium Pyrococcus furiosus, such as the protease shown in SEQ ID NO: 4 herein, or one having a sequence identity thereto of at least 90%, and wherein the protease has a thermostability value of more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% more than 100%, such as more that 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70.
  • the protease is a thermostable protease derived from the bacterium Pyrococcus furiosus, such as the protease shown in SEQ ID NO: 4 herein, or one having a sequence identity thereto of at least 95%, and wherein the protease has a thermostability value of more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% more than 100%, such as more that 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70.
  • the protease is a thermostable protease derived from the bacterium Pyrococcus furiosus, such as the protease shown in SEQ ID NO: 4 herein, or one having a sequence identity thereto of at least 99%, and wherein the protease has a thermostability value of more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% more than 100%, such as more that 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70.
  • step (a) When step (a) is carried out as a liquefaction step at high temperatures, i.e., above the initial gelatinization temperature, such as at temperatures between 80-90°C, such as around 85°C, the alpha-amylase may be a bacterial alpha-amylase.
  • the pH in step (a) is from 4-7, preferably 4.5-6.
  • a jet-cooking step is carried out before in step (a). Jet-cooking may be carried out at a temperature between 95-140°C for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes.
  • a process of the invention further comprises, before step (a), the steps of:
  • the bacterial alpha-amylase is derived from the genus Bacillus, such as a strain of Bacillus stearothermophilus, in particular a variant of a Bacillus stearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein, in particular a Bacillus stearothermophilus alpha-amylase truncated to have from 485-495 amino acids, such as around 491 amino acids.
  • the bacterial alpha-amylase is selected from the group of Bacillus stearothermophilus alpha-amylase variants comprising a double deletion, such as I181*+G182*, or I181*+G182*+N193F (using SEQ ID NO: 1 for numbering).
  • the bacterial alpha-amylase is selected from the group of Bacillus stearothermophilus alpha-amylase variants:
  • the parent Bacillus stearothermophilus alpha-amylase may be the one shown in SEQ ID NO: 1 or may be one having sequence identity thereto of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.
  • a Bacillus stearothermophilus alpha-amylase variant may be a variant of the one shown in SEQ ID NO: 1 or may be one having sequence identity thereto of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%.
  • the Bacillus stearothermophilus alpha-amylase variant has from 1-12 mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 mutations, compared to the parent alpha-amylase, especially the parent alpha-amylase shown in SEQ ID NO: 1.
  • Step (a) is followed by saccharification of dextrins in step (b).
  • a process of the invention may further comprise a pre-saccharification step, i.e., after step (a), but before saccharification step (b), carried out for 40-90 minutes at a temperature between 30-65°C.
  • saccharification is carried out at a temperature from 20-75°C, preferably from 40-70°C, such as around 60°C, and at a pH between 4 and 5.
  • fermentation step (c) or simultaneous saccharification and fermentation (SSF) are carried out carried out at a temperature from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around about 32°C.
  • fermentation step (c) or simultaneous saccharification and fermentation (SSF) i.e., combined steps (b) and (c) are ongoing for 6 to 120 hours, in particular 24 to 96 hours.
  • the starch-containing material converting step (a), saccharification step (b) and fermentation step (c) are carried out simultaneously or sequentially.
  • the starch-containing material converting step (a) is carried out at a temperature below the initial gelatinization temperature, preferably from 20-60°C, such as 25-40°C, such as around 30-34°C, such as around 32°C.
  • the starch-containing material is converted to dextrins and the dextrins are saccharified to a sugar by treating the starch-containing material with an alpha-amylase and glucoamylase below the initial gelatinization temperature of the starch-containing material.
  • the conversion of the starch-containing material to dextrins, the saccharification of the dextrins to sugars, and the fermentation of the sugars are carried out in a single step (i.e., raw starch hydrolysis step).
  • the process of the invention is carried out as a raw starch hydrolysis process (i.e., single step process or no-cook process)
  • the glucoamylase may preferably be derived from a strain of Trametes, such as a strain of Trametes cingulata, or a strain of Althelia, such as a strain of Athelia rolfsii.
  • Preferred alpha-amylases used in a raw starch hydrolysis process include alpha-amylases derived from a strain Rhizomucor, such as a strain of Rhizomucor pusillus, such as a Rhizomucor pusillus alpha-amylase with a starch-binding domain (SBD), such as a Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD.
  • a strain Rhizomucor such as a strain of Rhizomucor pusillus, such as a Rhizomucor pusillus alpha-amylase with a starch-binding domain (SBD), such as a Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD.
  • SBD starch-binding domain
  • the starch-containing material in raw starch hydrolysis processes are granular starch.
  • Said granular starch may be reduced the particle size, preferably by milling, to from 0.05 to 3.0 mm, preferably 0.1-0.5 mm.
  • the sugar level such as glucose level
  • the sugar level may be kept below 6 wt.-%, preferably below about 3 wt.-%, preferably below about 2 wt.-%, more preferred below about 1 wt.-%., even more preferred below about 0.5%, or even more preferred 0.25% wt.-%, such as below about 0.1 wt.-%.
  • the pH may be from 4-7, preferably 4.5-6.0, when conversion of the starch-containing material to dextrins, the saccharification of the dextrins to a sugar, and the fermentation of the sugar are carried out in a single step.
  • step (b) is carried out as a liquefaction step at a temperature above the gelatinization temperature
  • the carbohydrate-source generating enzyme used in step (b) is preferably a glucoamylase derived from Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably Trichoderma reesei; or a strain of Talaromyces , preferably Talaromyces emersonii, or a strain of Pycnoporus, or a strain of Gloephyllum.
  • the starch-containing material in step (a), including granular starch contains 20-55 wt.-% dry solids, preferably 25-40 wt.-% dry solids, more preferably 30-35% dry solids.
  • the protease is present in or added to the whole stillage in step (d) and/or the thin stillage, in step (e) or after separation in step (e), and/or the syrup in step (e').
  • Separation (i.e. dewatering) in step (e) may be carried out by centrifugation, preferably a decanter centrifuge, filtration, preferably using a filter press, a screw press, a plate-and-frame press, a gravity thickener or decker or any other separation technology known in the art.
  • the starch-containing material is cereal.
  • the starch-containing material is selected from the group consisting of corn, wheat, barley, cassava, sorghum, rye, potato, beans, milo, peas, rice, sago, sweet potatoes, tapioca, or any combination thereof.
  • the (desired) fermentation product may in an embodiment be selected from the group consisting of alcohols (e.g., ethanol, methanol, butanol, 1,3-propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2), and more complex compounds, including, for example, antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones.
  • the (desired) fermentation product is ethanol.
  • the desired fermentation product may be recovered by distillation.
  • oil may be recovered from the thin stillage and/or syrup/evaporated centrate, e.g., by extraction, such as hexane extraction.
  • the protease is derived from a strain of Pyrococcus, preferably a strain of Pyrococcus furiosus, such as the one shown in SEQ ID NO: 1 in US 6,258,726 or SEQ ID NO: 4 herein.
  • the protease has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO: 4 herein.
  • Separating whole stillage into thin stillage and wet cake in step (e), in order to remove a significant portion of the liquid/water may be done using any suitable separation technique, including centrifugation, pressing and filtration.
  • the separation/dewatering is carried out by centrifugation.
  • Preferred centrifuges in industry are decanter type centrifuges, preferably high-speed decanter type centrifuges.
  • An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval which is a high-performance decanter.
  • the separation is carried out using other conventional separation equipment such as a plate/frame filter presses, belt filter presses, screw presses, gravity thickeners and deckers, or similar equipment.
  • DDG "Distillers Dried Grains”
  • the wet cake is preferably dried under conditions that do not denature proteins in the wet cake.
  • the wet cake may be blended with syrup separated from the thin stillage and dried into DDG with Solubles (DDGS).
  • fermenting organisms used in step (c) for fermenting sugars in a fermentation medium into a desired fermentation product include fungal organisms, such as especially yeast.
  • Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.
  • the fermenting organism is added to the fermentation medium, so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 10 5 to 10 12 , preferably from 10 7 to 10 10 , especially about 5x10 7 .
  • yeast includes, e.g., RED STARTM and ETHANOL RED ⁇ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACCTM fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).
  • RED STARTM and ETHANOL RED ⁇ yeast available from Fermentis/Lesaffre, USA
  • FALI available from Fleischmann's Yeast, USA
  • SUPERSTART and THERMOSACCTM fresh yeast available from Ethanol Technology, WI, USA
  • BIOFERM AFT and XR available from NABC - North American Bioproducts Corporation, GA, USA
  • GERT STRAND available from Gert Strand AB, Sweden
  • FERMIOL available from DSM Specialties
  • starch-containing material Any suitable starch-containing material may be used according to the present invention.
  • the starting material is generally selected based on the desired fermentation product.
  • starch-containing materials suitable for use in a process of the invention, include whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixtures thereof or starches derived there from, or cereals. Contemplated are also waxy and non-waxy types of corn and barley.
  • Fermentation product means a product produced by a process including a fermentation step using a fermenting organism.
  • Fermentation products contemplated according to the invention include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones.
  • alcohols e.g., ethanol, methanol, butanol
  • organic acids e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid
  • ketones e.g.,
  • the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry.
  • Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • Preferred fermentation processes used include alcohol fermentation processes.
  • the fermentation product, such as ethanol, obtained according to the invention may preferably be used as fuel, that typically is blended with gasoline. However, in the case of ethanol it may also be used as potable ethanol.
  • the fermentation product such as ethanol may be separated from the fermentation medium, e.g., by distillation.
  • the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques.
  • the fermentation product may also be recovered by stripping or other method well known in the art.
  • One or more of the following enzyme activities may be used according to the invention.
  • thermostable protease (as defined herein) may be present or added during steps (d)-(e').
  • the protease may be of any origin as long as it has a thermostability value as defined herein of more than 20% and the Example 1.
  • the protease is of fungal origin.
  • the protease is of bacterial origin.
  • the protease has a thermostability of more than 50%, more than 60%, more than 70%, more than 80%, more than 90% more than 100%, such as more that 105%, such as more than 110%, such as more than 115%, such as more than 120% determined as Relative Activity at 80°C/70°C.
  • the protease has a thermostability value between 50 and 115%, such as between 50 and 70%, such as between 50 and 60%, such as between 100 and 120%, such as between 105 and 115% determined as Relative Activity at 80°C/70°C.
  • the protease is of bacterial origin.
  • the protease is derived from a strain of Pyrococcus, preferably a strain of Pyrococcus furiosus.
  • the protease is the one shown in SEQ ID NO: 1 in US 6,258,726 or SEQ ID NO: 4 herein.
  • the protease has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO: 1 in US 6,258,726 or SEQ ID NO: 4 herein.
  • the protease is present and/or added in an effective amount in step (a) or any of steps (d)-(e').
  • the protease is added in a concentration of 0.01-100, such 0.1-10 micro g/g DS.
  • the method of the invention may be carried out using any suitable alpha-amylase.
  • a bacterial alpha-amylase and/or a fungal alpha-amylase may be used.
  • the alpha-amylase is bacterial when step (a) is carried out as a liquefaction step at high temperatures, i.e., above the initial gelatinization temperature,
  • the alpha-amylase is fungal when step (a) is carried out at a temperature below the initial gelatinization temperature, such as when steps (a), (b) and (c) are carried out as a raw starch hydrolysis (single step process or no-cook process) as described above.
  • suitable bacterial alpha-amylases include the below mentioned.
  • Preferred bacterial alpha-amylases used in step (a) may be derived from a strain the genus Bacillus (sometimes referred to as Geobacillus ), including a strain of Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, or Bacillus subtilis.
  • Other bacterial alpha-amylases include alpha-amylase derived from a strain of the Bacillus sp.
  • NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375 all of which are described in detail in WO 95/26397 , and the alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31 .
  • the Bacillus alpha-amylase may also be a variant and/or hybrid, especially one described in any of WO 96/23873 , WO 96/23874 , WO 97/41213 , WO 99/19467 , WO 00/60059 , and WO 02/10355 .
  • WO 96/23873 WO 96/23874
  • WO 97/41213 WO 99/19467
  • WO 00/60059 WO 02/10355
  • Specifically contemplated alpha-amylase variants are disclosed in US patent nos. 6,093,562 , 6,297,038 or US patent no.
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants having a deletion of one or two amino acid in positions R179 to G182, preferably a double deletion disclosed in WO 1996/023873 - see e.g., page 20, lines 1-10, preferably corresponding to delta(181-182) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids R179 and G180 using SEQ ID NO:3 in WO 99/19467 for numbering.
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase
  • Bacillus alpha-amylases especially Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta(181-182) and further comprise a N193F substitution (also denoted I181* + G182* + N193F) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 .
  • Bacillus stearothermophilus alpha-amylase is one disclosed in WO 2011/082425 , such as one selected from the group of:
  • Bacillus stearothermophilus alpha-amylase has the following mutations: 181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (SEQ ID NO: 1).
  • the truncated Bacillus stearothermophilus alpha-amylase are typically naturally truncated to be about 491 amino acids long, such as from 485-495 amini acids long.
  • a hybrid alpha-amylase specifically contemplated comprises 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467 ) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467 ), with the following substitution: G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the numbering in SEQ ID NO: 4 in WO 99/19467 ).
  • variants having one or more of the mutations H154Y, A181T, N190F, A209V and Q264S and/or deletion of two residues between positions 176 and 179, preferably deletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO 99/19467 ).
  • bacterial alpha-amylase products and products containing alpha-amylases include TERMAMYLTM SC, LIQUOZYMETM SC, BAN (Novozymes A/S, Denmark) DEX-LOTM, SPEZYMETM XTRA, SPEZYMETM AA, SPEZYME FRED-L, SPEZYMETM ALPHA, GC358, SPEZYME RSL, SPEZYME HPA and SPEZYMETM DELTA AA (from DuPont, USA), FUELZYMETM (Verenium, USA).
  • a bacterial alpha-amylase may be added in step (a) in amounts as are well-known in the art.
  • the alpha-amylase activity is preferably present in an amount of 0.5-5,000 NU/g of DS, in an amount of 1-500 NU/g of DS, or more preferably in an amount of 5-1,000 NU/g of DS, such as 10-100 NU/g DS.
  • Fungal alpha-amylases (EC 3.2.1.1) are preferably of filamentous fungus origin.
  • the fungal alpha-amylase may be a fungal acid alpha-amylase.
  • Fungal acid alpha-amylases include acid alpha-amylases derived from a strain of the genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger alpha-amylases.
  • a preferred fungal alpha-amylase is a Fungamyl-like alpha-amylase which is preferably derived from a strain of Aspergillus oryzae.
  • the term "Fungamyl-like alpha-amylase” indicates an alpha-amylase which exhibits a high identity, i.e. more than 70%, more than 75%, more than 80%, more than 85% more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or even 100% identity to the mature part of the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874 .
  • Another preferred acid alpha-amylase is derived from a strain Aspergillus niger.
  • the acid fungal alpha-amylase is the one from A. niger disclosed as "AMYA_ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in more detail in WO 89/01969 (Example 3).
  • the acid Aspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes).
  • variants of said acid fungal amylase having at least 70% identity, such as at least 80% or even at least 90% identity, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 in WO 2004/080923 are contemplated.
  • a suitable commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark).
  • the fungal acid alpha-amylase may also be a wild-type enzyme comprising a carbohydrate-binding module (CBM) and an alpha-amylase catalytic domain (i.e., a none-hybrid), or a variant thereof.
  • CBM carbohydrate-binding module
  • alpha-amylase catalytic domain i.e., a none-hybrid
  • the wild-type acid fungal alpha-amylase is derived from a strain of Aspergillus kawachii.
  • compositions comprising fungal alpha-amylase include FUNGAMYLTM and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark).
  • the fungal acid alpha-amylase is a hybrid alpha-amylase.
  • Preferred examples of fungal hybrid alpha-amylases include the ones disclosed in WO 2005/003311 or U.S. Patent Publication no. 2005/0054071 (Novozymes) or US patent publication no. 2006/0148054 or US patent application no. 60/638,614 (Novozymes).
  • a hybrid alpha-amylase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate-binding domain/module (CBM), such as a starch binding domain, and optional a linker.
  • CD alpha-amylase catalytic domain
  • CBM carbohydrate-binding domain/module
  • contemplated hybrid alpha-amylases include those disclosed in Table 1 to 5 of the examples in co-pending US patent application no. 60/638,614 , including Fungamyl variant with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO: 2 herein and SEQ ID NO:100 in US 60/638,614 ), Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO: 3 herein and SEQ ID NO:101 in US 60/638,614 ), Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO:20 SEQ ID NO:72 and SEQ ID NO:96 in US application no.
  • An acid alpha-amylases may according to the invention be added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS.
  • Fungal alpha-amylases may be added to step (a) in a well know effective amount, preferably in the range from 0.001-1 mg enzyme protein per g DS (in whole stillage), preferably 0.01-0.5 mg enzyme protein per g DS.
  • a carbohydrate-source generating enzyme preferably a glucoamylase, may be present and/or added during saccharification step (b) or simultaneous saccharification and fermentation.
  • carbohydrate-source generating enzyme includes any enzymes generating fermentable sugars.
  • a carbohydrate-source generating enzyme is capable of producing a carbohydrate that can be used as an energy-source by the fermenting organism(s) in question, for instance, when used in a process of the invention for producing a fermentation product, such as ethanol.
  • the generated carbohydrates may be converted directly or indirectly to the desired fermentation product, preferably ethanol.
  • a mixture of carbohydrate-source generating enzymes may be used. Specific examples include glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators).
  • the carbohydrate-source generating enzyme is a glucoamylase.
  • the process of the invention may be carried out using any suitable glucoamylase.
  • the glucoamylase is of bacterial or fungal origin.
  • Contemplated glucoamylases include those from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase ( Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102 ), or variants thereof, such as those disclosed in WO 92/00381 , WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921 , A. oryzae glucoamylase ( Agric. Biol. Chem. (1991), 55 (4), p. 941-949 ), or variants or fragments thereof.
  • Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A ( Chen et al. (1996), Prot. Eng. 9, 499-505 ); D257E and D293E/Q ( Chen et al. (1995), Prot. Eng. 8, 575-582 ); N182 ( Chen et al. (1994), Biochem. J. 301, 275-281 ); disulphide bonds, A246C ( Fierobe et al. (1996), Biochemistry, 35, 8698-8704 ; and introduction of Pro residues in position A435 and S436 ( Li et al. (1997), Protein Eng. 10, 1199-1204 .
  • glucoamylases contemplated include glucoamylase derived from a strain of Athelia, preferably a strain of Athelia rolfsii (previously denoted Corticium rolfsii ) glucoamylase (see US patent no. 4,727,026 and ( Nagasaka,Y. et al.
  • the glucoamylase is derived from a strain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii.
  • the glucoamylase present and/or added during saccharification and/or fermentation is of fungal origin, preferably from a strain of Pycnoporus, or a strain of Gloephyllum.
  • the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ ID NO: 4 in WO 2011/066576 or SEQ ID NO: 18 herein.
  • the glucoamylase is derived from a strain of the genus Gloeophyllum , such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strain of Gloeophyllum as described in WO 2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16).
  • the glucoamylase is the Gloeophyllum sepiarium shown in SEQ ID NO: 2 in WO 2011/068803 or SEQ ID NO: 15 herein.
  • glucoamylases include glucoamylase derived from a strain of Trametes, preferably a strain of Trametes cingulata disclosed in WO 2006/069289 .
  • hybrid glucoamylase are contemplated according to the invention. Examples the hybrid glucoamylases disclosed in WO 2005/045018 . Specific examples include the hybrid glucoamylase disclosed in Table 1 and 4 of Example 1.
  • Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum ( EP 135,138 ), and C. thermohydrosulfuricum ( WO 86/01831 ).
  • compositions comprising glucoamylase include AMG 200L; AMG 300 L; SANTM SUPER, SANTM EXTRA L, SPIRIZYMETM PLUS, SPIRIZYMETM FUEL, SPIRIZYMETM B4U and AMGTM E (from Novozymes A/S); OPTIDEXTM 300 (from Genencor Int.); AMIGASETM and AMIGASETM PLUS (from DSM); G-ZYMETM G900, G-ZYMETM and G990 ZR (from Genencor Int.).
  • Glucoamylases may in an embodiment be added in an amount of 0.02-20 AGU/g DS, preferably 0.05-5 AGU/g DS (in whole stillage), especially between 0.1-2 AGU/g DS.
  • Glucoamylase may be added in an effective amount, preferably in the range from 0.001-1 mg enzyme protein per g DS, preferably 0.01-0.5 mg enzyme protein per g dry substrate.
  • LSCDS Bacillus stearothermophilus alpha-amylase with the mutations: I181*+G182*+N193F truncated to 491 amino acids (SEQ ID NO: 1 herein).
  • Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylase with the mutations: I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to 491 amino acids (SEQ ID NO: 1 herein).
  • TA Protease TA
  • TA Metallo protease derived from Thermoascus aurantiacus CGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 3 herein
  • TA 196 Metallo protease derived from Thermoascus aurantiacus CGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 3 herein with the following mutations: A27K+D79L +Y82F+S87G+D104P+A112P+A126V+D142L.
  • PF Protease PF
  • RH Protease RH
  • TF Protease TF
  • TF Protease derived from a filamentous fungus Thermobifida fusca shown in SEQ ID NO: 10 herein.
  • Glucoamylase SF is a glucoamylase derived from a strain of Talaromyces emersonii and is disclosed in WO9928448 and is available from Novozymes A/S.
  • Glucoamylase TC is a glucoamylase derived from Trametes cingulata disclosed in SEQ ID NO: 2 of WO 2006/069289 and available from Novozymes A/S.
  • Alpha-amylase JA is an alpha-amylase derived from Rhizomucor pusillus and disclosed disclosed as V039 in Table 5 in WO 2006/069290 .
  • Phadebas® tablets Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.
  • the measured 620 nm absorbance after 10 or 15 minutes of incubation is in the range of 0.2 to 2.0 absorbance units. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temperature, pH, reaction time, buffer conditions) 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
  • Alpha-amylase activity is alternatively determined by a method employing the PNP-G7 substrate.
  • PNP-G7 which is a abbreviation for p-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase.
  • Kits containing PNP-G7 substrate and alpha-glucosidase is manufactured by Bohringer-Mannheim (cat. No. 1054635).
  • BM 1442309 To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309).
  • BM 1462309 To prepare the alpha-glucosidase one bottle of alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309).
  • the working solution is made by mixing 5 ml alpha-glucosidase solution with 0.5 ml substrate.
  • the assay is performed by transforming 20 microL enzyme solution to a 96 well microtitre plate and incubating at 25°C. 200 microL working solution, 25°C is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 15 seconds over 3 minutes at OD 405 nm.
  • the slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions.
  • FAU Fungal Alpha-Amylase Unit
  • amount of enzyme which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6, Batch 9947275) per hour at Novozymes' standard method for determination of alpha-amylase based upon the following standard conditions: Substrate Soluble starch Temperature 37°C pH 4.7 Reaction time 7-20 minutes
  • Acid alpha-amylase activity is measured in AFAU ( A cid F ungal A lpha-amylase U nits), which are determined relative to an enzyme standard.
  • AMG 300 L wild type A. niger G1 AMG sold by Novozymes A/S.
  • the neutral alpha-amylase in this AMG falls after storage at room temperature for 3 weeks from approx. 1 FAU/mL to below 0.05 FAU/mL.
  • the acid alpha-amylase activity in this AMG standard is determined in accordance with AF 9 1/3 (Novo method for the determination of fungal alpha-amylase).
  • 1 AFAU is defined as the amount of enzyme, which degrades 5.260 mg starch dry matter per hour under standard conditions.
  • Iodine forms a blue complex with starch but not with its degradation products. The intensity of colour is therefore directly proportional to the concentration of starch.
  • Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under specified analytic conditions.
  • Alpha-amylase Starch + Iodine ⁇ Dextrins + Oligosaccharides 40°C, pH 2.5 Blue/violet t 23 sec.
  • Substrate starch, approx. 0.17 g/L
  • Buffer Citrate, approx.
  • Iodine (I 2 ) 0.03 g/L CaCl 2 : 1.85 mM pH: 2.50 ⁇ 0.05
  • Wavelength: Lambda 590nm
  • FAU-F F ungal Alpha- A mylase U nits ( F ungamyl) is measured relative to an enzyme standard of a declared strength. Reaction conditions Temperature 37°C pH 7.15 Wavelength 405 nm Reaction time 5 min Measuring time 2 min
  • KNU Alpha-amylase Activity
  • the alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.
  • KNU Kilo Novo alpha amylase Unit
  • the Novo Glucoamylase Unit is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37°C, pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.
  • An autoanalyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.
  • DMC Dimethyl casein
  • TNBS trinitrobenzene sul-phonic acid
  • the assay is further described in standard method document EB-SM-0218.02/02 available upon request from Novozymes A/S, Denmark.
  • Example 2 Thermostability in Example 2 is determined using the Zein-BCA assay.
  • Chemicals used were commercial products of at least reagent grade.
  • E. coli DH12S (available from Gibco BRL) was used for yeast plasmid rescue.
  • pJTP000 is a S. cerevisiae and E. coli shuttle vector under the control of TPI promoter, constructed from pJC039 described in WO 01/92502 , in which the Thermoascus aurantiacus M35 protease gene ( WO 03/048353 ) has been inserted.
  • Saccharomyces cerevisiae YNG318 competent cells MATa Dpep4[cir+] ura3-52, leu2-D2, his 4-539 was used for protease variants expression. It is described in J. Biol. Chem. 272(15): 9720-9727 (1997 ).
  • the solution is sterilized using a filter of a pore size of 0.20 micrometer.
  • Agar (2%) and H 2 O (approx. 761 mL) is autoclaved together, and the separately sterilized SC-glucose solution is added to the agar solution.
  • YPD Bacto peptone 20 g/l, yeast extract 10 g/L, 20 % glucose 100 mL/L.
  • PEG/LiAc solution 40 % PEG4000 50 ml, 5 M Lithium Acetate 1 mL.
  • Each well contains 200 microL of 0.05-0.1 % of zein (Sigma), 0.25 mM ZnSO 4 and 1 % of agar in 20 mM sodium acetate buffer, pH 4.5.
  • zein Sigma
  • 0.25 mM ZnSO 4 0.25 mM ZnSO 4
  • 1 % of agar in 20 mM sodium acetate buffer, pH 4.5.
  • Yeast transformation was performed using the lithium acetate method. 0.5 microL of vector (digested by restriction endnucleases) and 1 microL of PCR fragments is mixed. The DNA mixture, 100 microL of YNG318 competent cells, and 10 microL of YEAST MAKER carrier DNA (Clontech) is added to a 12 mL polypropylene tube (Falcon 2059). Add 0.6 mL PEG/LiAc solution and mix gently. Incubate for 30 min at 30°C, and 200 rpm followed by 30 min at 42°C (heat shock). Transfer to an eppendorf tube and centrifuge for 5 sec. Remove the supernatant and resolve in 3 mL of YPD.
  • E. coli transformation for DNA sequencing was carried out by electroporation (BIO-RAD Gene Pulser).
  • DNA Plasmids were prepared by alkaline method (Molecular Cloning, Cold Spring Harbor) or with the Qiagen® Plasmid Kit. DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNA Engine. The ABI PRISMTM 310 Genetic Analyzer was used for determination of all DNA sequences.
  • Themoascus M35 protease gene was amplified with the primer pair Prot F (SEQ ID NO: 5) and Prot R (SEQ ID NO: 6).
  • the resulting PCR fragments were introduced into S. cerevisiae YNG318 together with the pJC039 vector (described in WO 2001/92502 ) digested with restriction enzymes to remove the Humicola insolens cutinase gene.
  • Plasmid in yeast clones on SC-glucose plates was recovered to confirm the internal sequence and termed as pJTP001.
  • the primers AM34 (SEQ ID NO: 7) and AM35 (SEQ ID NO:8) were used to make DNA fragments containing any mutated fragments by the SOE method together with degenerated primers (AM34 + Reverse primer and AM35 + forward primer) or just to amplify a whole protease gene (AM34 + AM35).
  • PCR reaction system Conditions: 48.5 microL H 2 O 1 94°C 2 min 2 beads puRe Taq Ready-To-Go PCR (Amersham Biosciences) 2 94°C 30 sec 0.5 microL X 2 100 pmole/microL of primers 3 55°C 30 sec 0.5 microL template DNA 4 72°C 90 sec 2-4 25 cycles 5 72°C 10 min
  • DNA fragments were recovered from agarose gel by the Qiagen gel extraction Kit. The resulting purified fragments were mixed with the vector digest. The mixed solution was introduced into Saccharomyces cerevisiae to construct libraries or site-directed variants by in vivo recombination.
  • Yeast clones on SC-glucose were inoculated to a well of a 96-well micro titre plate containing YPD+Zn medium and cultivated at 28°C for 3 days.
  • the culture supernatants were applied to a 96-well zein micro titer plate and incubated at at least 2 temperatures (ex., 70°C and 80°C) for more than 4 hours or overnight.
  • the turbidity of zein in the plate was measured as A630 and the relative activity (higher/lower temperatures) was determined as an indicator of thermoactivity improvement.
  • the clones with higher relative activity than the parental variant were selected and the sequence was determined.
  • Yeast clones on SC-glucose were inoculated to a well of a 96-well micro titre plate and cultivated at 28°C for 3 days. Protease activity was measured at 65°C using azo-casein (Megazyme) after incubating the culture supernatant in 20 mM sodium acetate buffer, pH 4.5, for 10 min at a certain temperature (80°C or 84°C with 4°C as a reference) to determine the remaining activity. The clones with higher remaining activity than the parental variant were selected and the sequence was determined.
  • TCA trichloroacetic acid
  • the constructs comprising the protease variant genes were used to construct expression vectors for Aspergillus.
  • the Aspergillus expression vectors consist of an expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triose phosphate isomerase non translated leader sequence (Pna2/tpi) and the Aspergillus niger amyloglycosidase terminator (Tamg). Also present on the plasmid was the Aspergillus selective marker amdS from Aspergillus nidulans enabling growth on acetamide as sole nitrogen source.
  • the expression plasmids for protease variants were transformed into Aspergillus as described in Lassen et al., 2001, Appl. Environ. Microbiol. 67: 4701-4707 .
  • 10-20 strains were isolated, purified and cultivated in shake flasks.
  • Selected protease variants showing good thermostability were purified and the purified enzymes were used in a zein-BCA assay as described below. The remaining protease activity was determined at 60°C after incubation of the enzyme at elevated temperatures as indicated for 60 min.
  • Zein-BCA assay was performed to detect soluble protein quantification released from zein by variant proteases at various temperatures.
  • Enzyme doses Enzyme Dose Units 1 Control 0.00 ⁇ g ep/g DS 2 PF 20.00 ⁇ g ep/g DS 3 RH 20.00 ⁇ g ep/g DS 4 TA 20.00 ⁇ g ep/g DS 5 TA 196 20.00 ⁇ g ep/g DS 6 TF 20.00 ⁇ g ep/g DS
  • Tubes were centrifuged at 3,000 x g for 10 minutes in an Avanti JE Series centrifuge with JS-5.3 rotor (Beckmann Coulter). After centrifugation, the oil/hexane layer (supernatant) was removed using a positive displacement pipette (Ranin MR-250 and MR-1000), transferred to a pre-weighed 5 mL flip-top tube (Fisherbrand 03-338-1C), and reweighed. The density of the sample was measured using a Rudolph Research Analytical density meter (DDM 2911).
  • DDM 2911 Rudolph Research Analytical density meter

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Claims (6)

  1. Verfahren zum Gewinnen von Öl, umfassend
    (a) Umwandeln eines stärkehaltigen Materials zu Dextrinen mit einer alpha-Amylase
    (b) Verzuckern der Dextrine unter Verwendung eines eine Kohlenhydratquelle erzeugenden Enzyms, um einen Zucker zu bilden;
    (c) Fermentieren des Zuckers in einem Fermentationsmedium zu einem Fermentationsprodukt unter Verwendung eines fermentierenden Organismus;
    (d) Gewinnen des Fermentationsprodukts, um eine Schlempe zu bilden;
    (e) Trennen der Schlempe in Dünnschlempe und Dickschlempe;
    (e') gegebenenfalls Konzentrieren der Dünnschlempe zu Sirup;
    (f) Gewinnen von Öl aus der Dünnschlempe und/oder dem Sirup, wobei eine Protease mit mindestens 80% Identität zu SEQ ID NO: 4 und mit einem Thermostabilitätswert von mehr als 50%, bestimmt als relative Aktivität bei 80°C/70°C, vorliegt und/oder während Schritten (d)-(e') zugegeben wird.
  2. Verfahren nach Anspruch 1, wobei die Temperatur in Schritt (a) oberhalb der anfänglichen Geliertemperatur ist, wie bei einer Temperatur zwischen 80-90°C, wie um 85°C.
  3. Verfahren nach Anspruch 1 oder 2, wobei die Protease eine Thermostabilität von mehr als 60%, mehr als 70%, mehr als 80%, mehr als 90%, mehr als 100%, wie mehr als 105%, wie mehr als 110%, wie mehr als 115%, wie mehr als 120% aufweist, bestimmt als relative Aktivität bei 80°C/70°C.
  4. Verfahren nach einem beliebigen der Ansprüche 1-3, wobei die Protease aus einem Stamm von Pyrococcus, bevorzugt einem Stamm von Pyrococcus furiosus abgeleitet ist.
  5. Verfahren nach einem beliebigen der Ansprüche 1-4, wobei die Protease die hierin als SEQ ID NO: 4 gezeigte ist, oder wobei die Protease mindestens 85%, wie mindestens 90%, wie mindestens 95%, wie mindestens 96%, wie mindestens 97%, wie mindestens 98%, wie mindestens 99% Identität zu SEQ ID NO: 4 hierin aufweist.
  6. Verfahren nach einem beliebigen der Ansprüche 1-5, wobei die alpha-Amylase aus der Gattung Bacillus abgeleitet ist, wie einem Stamm von Bacillus stearothermophilus, insbesondere einer Variante einer Bacillus stearothermophilus alpha-Amylase wie der hierin als SEQ ID NO: 1 gezeigten, insbesondere ist die Bacillus stearothermophilus alpha-Amylase trunkiert, bevorzugt um von 485-495 Aminosäuren, wie um 491 Aminosäuren, aufzuweisen.
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